DEUBIQUITINASES IN CELL CYCLE CONTROL Project Summary Ubiquitin signaling contributes to virtually all aspects of cell physiology and is implicated in aging and disease. The covalent conjugation of polyubiquitin chains onto substrates triggers their degradation by the proteasome, as well as various other cellular outcomes. Ubiquitination is carried out by an enzymatic cascade of ubiquitin activators (E1), conjugators (E2) and ligases (E3). During normal cell cycles the ubiquitin system plays an essential and conserved role in remodeling the protein landscape. Ubiquitin substrates are determined by E3 ligases and much of our understanding of ubiquitin signaling has focused on the identity, substrates and mechanisms of E3s. However, ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed DUBs (deubiquitinases). Despite their critical role in sculpting the proteome, much less is known about the identity, substrates and mechanisms of DUBs in cell cycle progression, when compared to their E3 counterparts. Nevertheless, dysregulation of both E3s and DUBs alters cell cycle progression and has deleterious effects on genome integrity. Moreover, both E3s and DUBs can be perturbed in pathologies such as cancer, contributing to the biochemical and phenotypic features of disease. Thus, defining the identity, substrates and mechanisms of DUBs in cell cycle is essential to understanding how normal cell proliferation and genome stability are maintained and coordinated. We hypothesize that DUBs are essential for cell cycle progression and chromosome stability and are equally important as their E3 counterparts. We address this hypothesis in three specific aims, that combine complementary techniques, and which focus on the role of DUBs in major cell cycle transitions. In Aims 1 and 2 we investigate Cezanne/OTUD7B, an ovarian tumor family deubiquitinase, that we recently demonstrated is cell cycle regulated and which controls the M to G1 transition. In Aim 1, we will determine substrates for Cezanne using proteomics approaches, define mechanisms of DUB-substrate interactions, and the role of Cezanne in the degradation of substrates at M/G1. In Aim 2, we will expand this analysis to determine how Cezanne is itself regulated, both at the level of its abundance and activity, and then determine how these regulatory systems influence its role in cell division. Finally, in Aim 3, we determine the role of DUBs in a second major cell cycle transition, G1/S. The G1/S boundary is a major barrier to proliferation in normal and cancer cell cycles and relies heavily on ubiuqitin signaling. However, little is known about DUBs involved in G1/S. We will use computational approaches and loss-of-function screens, to identify and then investigate DUBs that control G1/S. Collectively, this proposal will fill significant knowledge gaps in the cell cycle, ubiquitin and DUB fields, related to roles and mechanisms of DUBs in proliferation and genome maintenance. My lab is uniquely positioned to address these questions, illustrated by our prior work, that includes global analysis of ubiquitination networks, detailed analysis of specific ubiquitin pathways and their role in cell cycle, and determination of the function and enzymology of cell cycle DUBs.